Endocrine mediation of vertebrate male alternative reproductive tactics: the next generation of studies

In many species of animals, males may achieve reproductive successvia one of several alternative reproductive tactics. Over thepast decade or so, there has been a concerted effort to investigateendocrine mechanisms that underlie such discrete behavioral(and often morphological) variation. In vertebrates, the firstgeneration of studies focused on potential organizational oractivational effects of steroid hormones (Moore, 1991; Mooreet al., 1998). Some of these studies have made it clear that,in addition to circulating hormone levels, one must also considerother aspects of the endocrine system, including hormone receptors,binding globulins and potential interactions among endocrineaxes. In this paper, I review recent work on endocrine mechanismsand suggest possibilities for future investigation. I highlighthow individual variation in sensitivity to environmental conditions,particularly with respect to various stressors, may accountfor the existence of alternative male reproductive phenotypes.Along these lines, I briefly explain the logic behind our workwith male phenotypes of longear sunfish (Lepomis megalotis)that is aimed at determining the tissue-specific distributionand activity of two enzymes that are common to androgen andglucocorticoid metabolism. A major goal of our work is to examinethe potential role of steroidogenic enzymes in the transductionof environmental information to influence the expression ofalternative male reproductive phenotypes.     

How integrated are behavioral and endocrine stress response traits? A repeated measures approach to testing the stress-coping style model

It is widely expected that physiological and behavioral stress responses will be integrated within divergent stress‐coping styles (SCS) and that these may represent opposite ends of a continuously varying reactive–proactive axis. If such a model is valid, then stress response traits should be repeatable and physiological and behavioral responses should also change in an integrated manner along a major axis of among‐individual variation. While there is some evidence of association between endocrine and behavioral stress response traits, few studies incorporate repeated observations of both. To test this model, we use a multivariate, repeated measures approach in a captive‐bred population of Xiphophorus birchmanni. We quantify among‐individual variation in behavioral stress response to an open field trial (OFT) with simulated predator attack (SPA) and measure waterborne steroid hormone levels (cortisol, 11‐ketotestosterone) before and after exposure. Under the mild stress stimulus (OFT), (multivariate) behavioral variation among individuals was consistent with a strong axis of personality (shy–bold) or coping style (reactive–proactive) variation. However, behavioral responses to a moderate stressor (SPA) were less repeatable, and robust statistical support for repeatable endocrine state over the full sampling period was limited to 11‐ketotestosterone. Although post hoc analysis suggested cortisol expression was repeatable over short time periods, qualitative relationships between behavior and glucocorticoid levels were counter to our a priori expectations. Thus, while our results clearly show among‐individual differences in behavioral and endocrine traits associated with stress response, the correlation structure between these is not consistent with a simple proactive–reactive axis of integrated stress‐coping style. Additionally, the low repeatability of cortisol suggests caution is warranted if single observations (or indeed repeat measures over short sampling periods) of glucocorticoid traits are used in ecological or evolutionary studies focussed at the individual level.     

Anxiety-related defensive behavioral responses in mice selectively bred for High and Low Activity

High and Low Activity strains of mice (displaying low and high anxiety-like behavior, respectively) with 7.8–20 fold differences in open-field activity were selected and subsequently inbred to use as a genetic model for studying anxiety-like behavior in mice (DeFries et al., 1978, Behavior Genetics, 8:3-13). These strains exhibited differences in other anxiety-related behaviors as assessed using the light–dark box, elevated plus-maze, mirror chamber, and elevated square-maze tests (Henderson et al., 2004, Behavior Genetics, 34: 267-293). The purpose of these experiments was three-fold. First, we repeated a 6-day behavioral battery using updated equipment and software to confirm the extreme differences in anxiety-like behaviors. Second, we tested novel object exploration, a measure of anxiety-like behavior that does not rely heavily on locomotion. Third, we conducted a home cage wheel running experiment to determine whether these strains differ in locomotor activity in a familiar, home cage environment. Our behavioral test battery confirmed extreme differences in multiple measures of anxiety-like behaviors. Furthermore, the novel object test demonstrated that the High Activity mice exhibited decreased anxiety-like behaviors (increased nose pokes) compared to Low Activity mice. Finally, male Low Activity mice ran nearly twice as far each day on running wheels compared to High Activity mice, while female High and Low Activity mice did not differ in wheel running. These results support the idea that the behavioral differences between High and Low Activity mice are likely to be due to anxiety-related factors and not simply generalized differences in locomotor activity.     

Maternal condition reduces fear behaviors but not the endocrine response to an emotional threat in virgin female rats

Lactating dams and maternal virgin females are less fearful in behavioral tests compared with non-maternal animals, suggesting that maternal condition per se reduces the negative value of threatening stimuli. In addition, lactating females exhibit a diminished hypothalamic-pituitary-adrenal response to potential environmental threats. Can the maternal condition, independently of the endocrine profile of lactation, promote a reduction in the behavioral as well as in the endocrine response to an emotional stressor? To answer this question, anxiety-related and fear behaviors as well as the levels of corticosterone were evaluated in response to a bright-lit open field-loud noise model in maternal and non-maternal non-ovariectomized virgin females and lactating dams in the presence of the pups. Maternal animals, both lactating and virgin, presented an increased exploration of the bright-lit open field and a significant reduction of fear behaviors, indicated by the decreased flight and immobility responses to the subsequent activation of a loud noise, in comparison to non-maternal virgins. Interestingly, maternal virgin females, as non-maternal rats, showed high corticosterone plasma levels, in contrast to the lower endocrine response exhibited by lactating dams when confronted to this threat. Present results suggest that maternal condition allows females to take risks when caring for their young, a behavioral strategy that is independent of the reduced hypothalamic-pituitary-adrenal axis response characteristic of lactation. This evidence points towards a clear dissociation in the mechanisms regulating behavioral and endocrine responses to emotional stressors during motherhood.     

Sex-specific responses to urinary chemicals by the mouse vomeronasal organ

Social behaviors of most mammals are affected by chemical signals, pheromones, exchanged between conspecifics. Previous experiments have shown that behavioral responses to the same pheromone differ depending on the sex and endocrine status of the respondent. Although the exact mechanism of this dimorphism is not known, one possible contributor may be due to sexually dimorphic receptors or due to differences in central processing within the brain. In order to investigate the differences in response between male and female mice to the same pheromonal stimulus two urinary compounds (2-heptanone and 2,5-dimethylpyrazine) were used to stimulate the production of Inositol (1,4,5)-trisphosphate (IP(3)) in microvillar membrane preparations of the vomeronasal organ as an indirect measurement of pheromonal stimulation. Incubation of such membranes from prepubertal mice with urine from the same sex or opposite sex, results in an increase in production of IP(3). This stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore we found that 2-heptanone present in both male and female urine was capable of stimulating increased production of IP(3) in the female VNO but not the male VNO. Finally, 2,5-dimethylpyrazine present only in female urine was also only capable of stimulating increased production of IP(3) in the female VNO.     

Fmr1 knockout mice are impaired in a leverpress escape/avoidance task

Fragile X syndrome (FXS) is the most common form of inherited mental retardation (MR). FXS is typically caused by a mutation of the Fmr1 gene (Verkerk et al. 1991, Cell 65, 905-914). To better understand the role of Fmr1 and its gene product fragile X mental-retardation protein (FMRP) in central nervous system function, researchers have turned to the use of animal model systems to generate an Fmr1 knockout (KO) mouse that is deficient in FMRP (Bakker et al. 1994, Cell 78, 23-33). Unfortunately, a number of studies have found no consistent, robust learning and memory impairment in the Fmr1 KO mice. We conducted a study to assess the performance of Fmr1 KO and wildtype (WT) animals in a leverpress escape/avoidance paradigm. Fmr1 KO and WT littermates were studied in four daily 1-h sessions. The Fmr1 KO mice performed fewer avoidance and total responses than WT mice. The KO animals were not simply deficient in avoidance, but a within-factor ANOVA revealed that they did not acquire the leverpress response to any appreciable degree. Observation during the sessions indicated that the Fmr1 KO animals clearly responded to the shock, eliminating an obvious sensory explanation for the deficit. The fact that other studies have found that the KO mice displayed increased exploratory and locomotor activity compared with WT controls argues against a motoric deficit. Future studies will attempt to delineate the nature of the behavioral deficit as well as attempt to rescue the response with glutamatergic or dopaminergic agents.     

Wild mice provide insights into natural killer cell maturation and memory

The immune system has evolved, and continues to evolve, in response to the selection pressure that infections exert on animals in their natural environments, yet much of our understanding about how the immune system functions comes from studies of model species maintained in the almost complete absence of such environmental selection. The scientific discipline of immunology has among its aims the improvement of human and animal health by the application of immunological knowledge. As research on humans and domesticated animals is highly constrained-ethically, logistically and financially-experimental animal models have become an invaluable tool for dissecting the functioning of the immune system. The house mouse (Mus musculus) is by far the most widely used animal model in immunological research but laboratory-reared mice provide a very narrow view of the immune system-that of a well-fed and comfortably housed animal with minimal exposure to microbial pathogens. Indeed, so much of our immunological knowledge comes from studies of a very few highly inbred mouse strains that-to all intents and purposes-our immunological knowledge is based on enormously detailed studies of very small numbers of individual mice. The limitations of studies in inbred strains of laboratory mice are well-recognized (Pedersen & Babayan 2011), but serious attempts to address these limitations have been few and far between. However, the emerging field of 'ecological immunology' where free-living populations are studied in their natural habitat is beginning to redress this imbalance (Viney et al. 2005; Martin et al. 2006; Owen et al. 2010; Abolins et al. 2011). As demonstrated in the work by Boysen et al. (2011) in this issue of Molecular Ecology, studies in wild animal populations-especially free-living M. musculus-represent a valuable bridge between studies in humans and livestock and studies of captive animals.     

细胞色素P450酶的结构、功能与应用研究进展

细胞色素P450 (cytochrome P450,CYP)酶是广泛存在于微生物、动植物及人体中与膜结合的血红蛋白类酶,具有氧化、环氧化、羟化、去甲基化等多种生物催化活性。CYP酶在药物、类固醇、脂溶性维生素和许多其他类型化学物质的代谢中具有重要作用,其在异源物质的解毒、药物相互作用和内分泌功能等领域的研究是热点问题。本综述对CYP的结构、功能、临床应用与开发前景进行了概述,并对其最新的研究现状和发展前景进行探讨。     

Stimulation of maternal responsiveness after pregnancy termination in rats: Effect of time of onset of behavioral testing

To assess the effects of varying the time interval between surgical pregnancy termination and onset of behavioral testing on the expression of maternal behavior, both responsiveness to foster young and nest building-were measured in rats ovariectomized (O), hysterectomized (H), ovariectomized and hysterectomized (OH), or sham-operated (I) on Day 17 of pregnancy at 10:00 and first tested for maternal responsiveness 6, 24, or 48 hr after surgery. When behavioral testing was started 6 hr after surgery O and OH groups responded maternally to foster pups faster than H or I groups, and H females responded maternally faster than I animals (O = OH < H < I). In animals first tested 24 hr after surgery shorter latencies to retrieve and group foster young and nest build were found in the three pregnancy-terminated groups than in I animals, while the three pregnancy-terminated groups did not differ from one another with respect to either behavioral measure (O = OH = H < I). In contrast, when testing was initiated 48 hr after surgery, hysterectomized (H) animals responded maternally faster than did ovariectomized (O and OH) or intact pregnant (I) groups (H < OH < I, H < O = I). These data demonstrate that varying the time of onset of behavioral testing after surgical pregnancy termination affects elicitation of responsiveness to foster young and also affects nest building behavior in pregnancy-terminated animals. The differences in onset of maternal behaviors among pregnancy-terminated animals are discussed in relation to progesterone and estrogen secretion after surgery.     

Effect of enrichment on variation and results in the light/dark test

Several confounding factors may influence the outcome of an experiment and the extent of inter-individual variation. The aim of this study was to investigate if cage enrichment induces an effect on experimental mean values and on inter-individual variation in the light/dark paradigm using diazepam as the anxiolytic drug. The behaviour of 216 naive adult male mice of two different strains (BALB/c and C57BL/6) was studied. The animals were housed in groups of four in 'non-enriched', 'enriched' (nesting material) or 'super-enriched' (nest-box, nesting material, wooden gnawing stick and PVC tube) cages. After 5 weeks the animals were assigned to one of three treatments: control (no injection), sham (saline injection i.p.) or diazepam (1 mg/kg bw i.p.) and tested in the light/dark test for 5 min. Variation data were analysed using three different methods (mean absolute deviation, coefficient of variation and power analysis). The C57BL/6 mice scored higher than BALB/c mice in activity related measurements and showed a less 'emotional' behaviour profile in the pharmacological control situation of the light/dark test. In this study the anxiolytic effect of diazepam was clear in BALB/c mice but absent in C57BL/6 mice. Mice housed in enriched and super-enriched cages gained more weight than mice in non-enriched cages, although food intake was not affected. Generally, the strain of mouse had the greatest impact on both mean values and variation. However, there was no consistent increase for one particular strain. The choice of statistical method for analysing variation may influence the interpretation of within-group variability, but none of the methods showed any significant differences between standard and enriched conditions on variability in any of the parameters measured.     

The effect of teratogens on maternal corticosterone levels and cleft incidence in A/J mice.

It is unknown whether orofacial clefting, one consequence of teratogenic exposure, results from a direct interaction between the teratogen and the embryonic palate, or indirectly from maternal alterations caused by the teratogen. In the current study pregnant A/J mice were exposed to one of three cleft-inducing agents in order to examine the relationship between drug-induced clefting and the response of maternal plasma corticosterone to drug administration. The agents used, haloperidol (HAL), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or phenytoin (PHT), were administered in teratogenic doses between 0800 and 0930 on gestational day 10 (GD 10). For corticosterone determinations, mice were dosed on GD 10, and blood was collected at 1, 4, 24, or 48 hr after dosing. For fetal evaluation of cleft lip and/or cleft palate, mice were dosed on GD 10 and killed on GD 18. Phenytoin was the most potent inducer of cleft lip and palate and induced a sustained elevation of plasma corticosterone in maternal animals. The other treatments, in order of decreasing potency to induce clefting and/or cause an elevation of corticosterone in plasma were 2,4,5-T > HAL > controls. Correlations between maternal corticosterone levels and clefting incidence were very high at all time points examined; total exposure (area under the curve) was also highly correlated. A linear relationship between drug-induced increases in maternal corticosterone levels and the incidence of clefting in A/J mice was evident. Based on these findings, we believe that increased maternal corticosterone levels may play a role in orofacial clefting in A/J mice.     

Reduction of animal usage by serial bleeding of mice for pharmacokinetic studies: application of robotic sample preparation and fast liquid chromatography–mass spectrometry

Typically, pharmacokinetic studies in mice require one animal per time point, thus resulting in differences due to dosing error, animal to animal variation and more importantly the euthanasia of a large number of animals. A method for the determination of pharmacokinetic data from serially bled mice to support early drug discovery is described. Sample analysis relies on liquid chromatography coupled with tandem mass spectrometry permitting robust and reproducible analysis requiring approximately 3 min per sample. Several parameters are discussed including the method of sample collection, preparation and analysis. The use of serially bled mice has lead to a remarkable reduction in animal usage and a corresponding reduction in compound required for such experiments. Using conventional methodology, a nine-point pharmacokinetic curve with four animals per time point would require 36 mice. With the method described below, only four mice in total are used and euthanasia is not required, permitting reuse after several weeks recovery and washout. Also, pharmacodynamic–pharmacokinetic correlation is possible and is demonstrated using a mouse model of diabetes.     

Maternal and fetal antibrain antibodies in development and disease

Recent evidence has emerged indicating that the maternal immune response can have a substantial deleterious impact on prenatal development (Croen et al., [2008]: Biol Psychiatry 64:583-588). The maternal immune response is largely sequestered from the fetus. Maternal antibodies, specifically immunoglobulin G (IgG), are passed to the fetus to provide passive immunity throughout much of pregnancy. However, both protective and pathogenic autoantibodies have equal access to the fetus (Goines and Van de Water [2010]: Curr Opin Neurol 23:111-117). If the mother has an underlying autoimmune disease or has reactivity to fetal antigens, autoantibodies produced before or during pregnancy can target tissues in the developing fetus. One such tissue is the fetal brain. The blood brainbarrier (BBB) is developing during the fetal period allowing maternal antibodies to have direct access to the brain during gestation (Diamond et al. [2009]: Nat Rev Immunol; Braunschweig et al. [2011]; Neurotoxicology 29:226-231). It has been proposed that brain injury by circulating brain-specific maternal autoantibodies might underlie multiple congenital, developmental disorders (Lee et al. [2009]: Nat Med 15:91-96). In this review, we will discuss the current state of research in the area of maternal autoantibodies and the development of autism. ? 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012.     

The genetic basis of inbreeding avoidance in house mice

Animals might be able to use highly polymorphic genetic markers to recognize very close relatives and avoid inbreeding. The major histocompatibility complex (MHC) is thought to provide such a marker because it influences individual scent in a broad range of vertebrates. However, direct evidence is very limited. In house mice (Mus musculus domesticus), the major urinary protein (MUP) gene cluster provides another highly polymorphic scent signal of genetic identity that could underlie kin recognition. We demonstrate that wild mice breeding freely in seminatural enclosures show no avoidance of mates with the same MHC genotype when genome-wide similarity is controlled. Instead, inbreeding avoidance is fully explained by a strong deficit in successful matings between mice sharing both MUP haplotypes. Single haplotype sharing is not a good guide to the identification of full sibs, and there was no evidence of behavioral imprinting on maternal MHC or MUP haplotypes. This study, the first to examine wild animals with normal variation in MHC, MUP, and genetic background, demonstrates that mice use self-referent matching of a species-specific polymorphic signal to avoid inbreeding. Recognition of close kin as unsuitable mates might be more variable across species than a generic vertebrate-wide ability to avoid inbreeding based on MHC.     

Do Sex Differences in the Brain Explain Sex Differences in the Hormonal Induction of Reproductive Behavior? What 25 Years of Research on the Japanese Quail Tells Us

Early workers interested in the mechanisms mediating sex differences in morphology and behavior assumed that differences in behavior that are commonly observed between males and females result from the sex specificity of androgens and estrogens. Androgens were thought to facilitate male-typical traits, and estrogens were thought to facilitate female-typical traits. By the mid-20th century, however, it was apparent that administering androgens to females or estrogens to males was not always effective in sex-reversing behavior and that in some cases a “female” hormone such as an estrogen could produce male-typical behavior and an androgen could induce female-typical behavior. These conceptual difficulties were resolved to a large extent by the seminal paper of C. H. Phoenix, R. W. Goy, A. A. Gerall, and W. C. Young in (1959,Endocrinology65, 369–382) that illustrated that several aspects of sexual behavior are different between males and females because the sexes have been exposed during their perinatal life to a different endocrine milieu that has irreversibly modified their response to steroids in adulthood. Phoenixet al.(1959) therefore formalized a clear dichotomy between the organizational and activational effects of sex steroid hormones. Since this paper, a substantial amount of research has been carried out in an attempt to identify the aspects of brain morphology or neurochemistry that differentiate under the embryonic/neonatal effects of steroids and are responsible for the different behavioral response of males and females to the activation by steroids in adulthood. During the past 25 years, research in behavioral neuroendocrinology has identified many sex differences in brain morphology or neurochemistry; however many of these sex differences disappear when male and female subjects are placed in similar endocrine conditions (e.g., are gonadectomized and treated with the same amount of steroids) so that these differences appear to be of an activational nature and cannot therefore explain sex differences in behavior that are still present in gonadectomized steroid-treated adults. This research has also revealed many aspects of brain morphology and chemistry that are markedly affected by steroids in adulthood and are thought to mediate the activation of behavior at the central level. It has been explicitly, or in some cases, implicitly assumed that the sexual differentiation of brain and behavior driven by early exposure to steroids concerns primarily those neuroanatomical/neurochemical characteristics that are altered by steroids in adulthood and presumably mediate the activation of behavior. Extensive efforts to identify these sexually differentiated brain characteristics over the past 20 years has only met with limited success, however. As regards reproductive behavior, in all model species that have been studied it is still impossible to identify satisfactorily brain characteristics that differentiate under early steroid action and explain the sex differences in behavioral activating effects of steroids. This problem is illustrated by research conducted on Japanese quail (Coturnix japonica), an avian model system that displays prominent sex differences in the sexual behavioral response to testosterone, and in which the endocrine mechanisms that control sexual differentiation of behavior have been clearly identified so that subjects with a fully sex-reversed behavioral phenotype can be easily produced. In this species, studies of sex differences in the neural substrate mediating the action of steroids in the brain, including the activity of the enzymes that metabolize steroids such as aromatase and the distribution of steroid hormone receptors as well as related neurotransmitter systems, did not result in a satisfactory explanation of sex differences in the behavioral effectiveness of testosterone. Possible explanations for the relative failure to identify the organized brain characteristics responsible for behavioral sex differences in the responsiveness to steroids are presented. It is argued that novel research strategies may have to be employed to successfully attack the fundamental question of the hormonal mechanisms regulating sex differences in behavior.     

Developmental exposure to low-dose estrogenic endocrine disruptors alters sex differences in exploration and emotional responses in mice

Estrogenic endocrine disruptors (EEDs) are naturally occurring or man-made compounds present in the environment that are able to bind to estrogen receptors and interfere with normal cellular development in target organs and tissues. There is mounting evidence that EEDs can interfere with the processes of sexual differentiation of brain and behavior in different animal models. We investigated the effects of maternal exposure to EEDs, at concentrations within the range of human exposure and not patently teratogenic, on behavioral responses of male and female house mice (Mus musculus domesticus) before and after puberty. Pregnant dams were trained to spontaneously drink daily doses of corn oil with or without the estrogenic plastic derivative, bisphenol A (BPA 10 microg/kg), or the estrogenic insecticide methoxychlor (MXC 20 microg/kg) from gestation day 11 to postpartum day 8. Their male and female offspring were examined at different ages to examine several components of explorative and emotional behaviors in 3 experimental paradigms: a novelty test before puberty and, as adults, a free-exploratory open-field test and the elevated plus maze test. The main results are sex differences in control mice on a number of behavioral responses at both ages and in all experimental paradigms, while perinatal exposure to BPA or MXC decreased or eliminated such sex differences. The present findings are evidence of long-term consequences of developmental exposure to BPA and MXC on neurobehavioral development and suggest a differential effect of low-dose exposure to these estrogenic chemicals in males and females.     

Cohesin and the maternal age effect

During meiosis in human oocytes, chromosome nondisjunction increases with maternal age, leading to disorders such as Down's syndrome. In a recent study in Nature Genetics, Hodges et al. (2005) show that mice with a mutation in the meiosis-specific cohesin protein SMC1beta exhibit age-dependent defects in meiosis. These defects are similar to those observed in oocytes of older human mothers. Their results implicate an age-dependent loss of function in SMC1beta (or related proteins) in the maternal age effect of humans.     

Characterization and Social Correlates of Fecal Testosterone and Cortisol Excretion in Wild Male <Emphasis Type="Italic">Saguinus mystax</Emphasis>

Reproductive success in male primates can be influenced by testosterone (T) and cortisol (C). We examined them in wild Saguinus mystax via fecal hormone analysis. Firstly, we wanted to characterize male hormonal status over the course of the year. Further we tested the influence of the reproductive status of the breeding female, social instability, and intergroup encounter rates on T levels, comparing the results with predictions of the challenge hypothesis (Wingfield et al., 1990). We also tested for interindividual differences in hormonal levels, possibly related to social or breeding status. We collected data during a 12-mo study on 2 groups of moustached tamarins at the Estación Biológica Quebrada Blanco in northeastern Peru. We found fairly similar T and C levels over the course of the year for all males. Yet an elevation of T shortly after the birth of infants, during the phase of ovarian inactivity of the groups breeding female, was evident. Hormonal levels were not significantly elevated during a phase of social instability, did not correlate with intergroup encounter rates, and did not differ between breeding and nonbreeding males. Our results confirm the challenge hypothesis (Wingfield et al., 1990). The data suggest that reproductive competition in moustached tamarins is not based on endocrinological, but instead on behavioral mechanisms, possibly combined with sperm competition.     

Response to Midgley: the costs of reproduction cannot differ between the sexes

Many studies have compared the reproductive cost and vegetative growth at a particular time point. In our review (Liu et al. 2021b), we summarized those results but did not compare absolute reproductive costs between the sexes (Hultine et al. 2016; Juvany and Munné-Bosch 2016). Moreover, we did not propose that the observed vegetative and environmental differences between the sexes were the only reasons for differences in sexual functioning, especially in the spreading and receiving of pollen (Midgley 2022). Yet, we need further evidence to support the argument. Previous studies have shown that differences in primary and physiological traits between the sexes strongly depend on the plant species and their environmental conditions, and that they may arise from a number of reasons, such as differences in trait optima of each sex along a series of resource gradients, sexual selection and sex-specific responses to sexual selection (Barrett and Josh 2013; Geber et al. 1999; Juvany and Munné-Bosch 2016; Kohorn et al. 1994; Rabska et al. 2021; Retuerto et al. 2018; Scopece et al. 2021; Wang et al. 2021). In the comment, Midgley (2022) stated that our general argument is that ‘the net reproductive costs are higher for females because they not only flower but must also produce fruits/cones/seeds (Figure 3). Midgley (2022) suggests (Figure 2) that females can ameliorate their higher costs of reproduction by maximizing resource acquisition and resource gain’. However, in our review, we summarized the general opinion and pointed out that this pattern was not universal (see more detail in Liu et al. 2021b). In consistent with previous reviews, our review argues that there is no widespread rule in sex-related differences in the cost of reproduction despite the general opinion that females have higher reproductive costs than males (Darwin 1877; Liu et al. 2021b; Lloyd and Webb 1977). We summarized possible factors causing biased sex ratios in plants, rather than only underpinning the higher net reproductive costs in females than in males (Liu et al. 2021b). Similarly, we proposed possible mechanisms causing sexual differences in responses to biotic stress, rather than underpinning the higher net reproductive costs in females than in males (Liu et al. 2021b), which is also adapted from Núñez-Farfán and Valverde (2020).